ABSTRACT During the last funding interval as a merit award, this grant focused on the T cell receptor (TCR) complex composed of an [unreadable][unreadable] heterodimer and non-covalently associated CD3 signaling components. We continued crystallographic analysis with TCRs, providing the first structure of an [unreadable][unreadable] heterodimer in complex with a peptide/MHC (pMHC) class II ligand. In conjunction with a crystallographic structure of CD4 D1D2 binding to the same MHC class II molecule, a hydrophobic concavity formed by residues from membrane proximal [unreadable]2 and [unreadable]2 MHC class II domains was revealed. We excluded a direct TCRab-CD4 interaction, instead revealing how TCR[unreadable][unreadable] and CD4 signaling is coordinated in a "V-shape" around the antigenic peptide/pMHC class II complex. Solution structures of CD3[unreadable][unreadable] and CD3[unreadable][unreadable] ectodomain complexes were determined by NMR, uncovering for each dimer a unique side-to-side hydrophobic interface between their two Ig-like domains with parallel pairing of respective C-terminal [unreadable]-strands, and suggesting how rigidified CD3 elements participate in TCR-based signal transduction. To now characterize the structural basis for early signal transduction events via this TCR[unreadable][unreadable] complex, four aims are proposed involving the CD3[unreadable][unreadable] heterodimer and associated TCR [unreadable] chain. First, NMR-based methods will be used to determine the precise binding site on CD3[unreadable][unreadable] of activating and non-activating anti-CD3[unreadable] mAbs, given equivalent binding affinities. Second, CD3[unreadable][unreadable]-associated TCR[unreadable] chain recognition function during thymic development and T cell activation accounting for V[unreadable] repertoire bias will be assessed. Third, the structure and function of the transmembrane (TM) segments of CD3[unreadable] and [unreadable] will be determined and their interaction with that of TCR [unreadable] ascertained. Fourth, electron paramagnetic resonance (EPR) methods will be exploited to define the orientation and disposition of TM elements and how pMHC or anti-CD3[unreadable] mAbs affect TM depth, orientation and structural conformation. Distance measurements between the [unreadable] chain and CD3[unreadable] and CD3[unreadable] ectodomains will also be examined before and after pMHC or anti- CD3 mAb ligation. That orthological rather than vertical force via CD3 components activates T cells suggests a dynamic mechanosensor TCR model;pMHC pulls on the TCR from the opposing antigen-presenting cell surface such that the [unreadable][unreadable] heterodimer then presses on the CD3 ectodomains to initiate signaling.